Radioimmunoassay For Growth Hormone Research Articles

Possible evidence for the existence of a growth hormone fragment in porcine pituitary.

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126-180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of 125I-labelled fragment B by procine growth hormone required a 10(3) M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of 125I-labelled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possible representing a growth hormone fragment.

Purification and properties of teleost growth hormone

Highly purified growth hormone (GH) has been prepared from the pituitary glands of a euryhaline teleost, Tilapia mosambica. Tilapia GH was obtained in a yield of 1400 mg/kg wet weight tissue. It was found to have a molecular weight (gel filtration) of 22,200 daltons, a sedimentation coefficient ( s 20,w) of 2.19, and an α-helix content (circular dichroism) of 50%. Isoleucine was found to be the major amino-terminal residue; leucine was found to be COOH terminal. The amino acid composition, disc gel electrophoresis pattern, and circular dichroism spectra were similar to those of mammalian GHs. Tilapia GH was found to have a low but significant activity in the rat tibia assay and showed immunological relatedness to mammalian GH in a rat GH radioimmunoassay. Antiserum was prepared against the Tilapia GH and characterized in agar diffusion experiments and radioimmunoassay. Results from these investigations demonstrated a significant degree of cross-reaction between Tilapia GH and pituitary extract from another teleost (perch), but purified tetrapod GHs were essentially nonreactive. The data indicate a significant resemblance between Tilapia GH and mammalian GHs and suggest that the GH structure has been strongly conserved during evolution.

Tyrosylated analogues of somatostatin.

The total synthesis of [tyr1]-, [tyr6]-, E1Tyr7], [d-tyr8]-, [tyr11]-,and [tyr1,D-Trp8]somatostatin is described. The biological potencies of these analogues in vitro (inhibition of spontaneous secretion of radioimmunoassayable growth hormone by cultured rat anterior pituitary cells) as compared with that of somatostatin (100) were found to be 116, 29, 108, 10, 65, and 400, respectively; in vivo, in the rat, these peptides were assayed for their ability to inhibit the spontaneous release of insulin and glucagon. No statistically significant discrepancies were observed in the potency values obtained in vitro and in vivo further indicating similarities of specificities and sensitivities of the pituitary and pancreatotropic receptors.

Effect of rat stalk median eminence extracts on rat GH secretion in vivo.

Estradiol (0.2 mug), injected subcutaneously for 10 days to adult male rats, increased plasma growth hormone (GH) levels as compared with oil-treated controls. In estradiol-pretreated (10 days), urethane-anesthetized rats, the first as well as the second of two successive intracarotid injections, at 1-hour intervals, of one rat stalk median eminence equivalent evoked a significant rise in plasma radioimmunoassayable GH. Under the same conditions, cerebral cortex extracts (1 equivalent) induced a slight elevation whereas vasopressin (30 mU) or serotonin (200 ng) were ineffective. These results indicate that estrogen-primed, urethane-anesthetized rats can be used to demonstrate GH-releasing activity in rat SME extracts.

Growth hormone and cortisol levels in the annual cycle of white-tailed deer (Odocoileus virginianus).

The annual cycles of growth hormone and cortisol levels in white-tailed deer were followed. No significant difference was found in the average cortisol levels in a large group of animals during the year. In contrast to cortisol, growth hormone (GH) investigated by heterologous bovine GH radioimmunoassay, seems to have a distinct annual rhythm. High levels of GH in plasma were found between casting and the first week of antler growth with the peak of 23.1 ng/ml (estimated in bovine GH equivalents) in the middle of April. From the middle of June until the middle of November a plateau averaging 3 ng/ml was found. The relationships between very rapid growth of a bone tissue and the influence of GH and sex hormones are discussed.

Isolation of somatostatin (a somatotropin release inhibiting factor) of ovine hypothalamic origin.

Isolation of somatostatin, a tetradecapeptide of ovine origin inhibiting somatotropin secretion, is reported. About 490 000 hypothalamic fragments were submitted to alcohol–chloroform extraction, countercurrent distribution, ion-exchange chromatography, gel filtration, and partition chromatography. Of the 8.5 mg material thus obtained, 77% was accounted for by a peptide shown homogeneous by electrophoresis, thin-layer chromatography, and amino acid analysis. The peptide inhibits the secretion of radioimmunoassayable growth hormone at doses of ≥ 1.0 nM in vitro and 400 ng per rate in vivo.

Altered growth hormone homeostasis during acute bacterial sepsis in the rhesus monkey.

ABSTRACT An impressive hypersecretion of growth hormone (GH) occurs in response to an intravenous (iv) glucose load during the febrile phase of a viral infection (sandfly fever) in man. To study the mechanism of such hypersecretion using an appropriate animal model, conscious male rhesus monkeys were given iv glucose tolerance tests (GTT) (0.5 g/kg) on a control day (GTT-1) and at the height of illness (GTT-2) 24 hr after iv inoculation with 108 virulent Diplococcus pneumoniae (n = 9) or 109 virulent Salmonella typhimurium (n = 5). Sequential plasma samples were obtained from femoral vein catheters for radioimmunoassayable GH, glucose, and free fatty acids (FFA). There was no glucose intolerance or FFA abnormalities during GTT-2 in either group. An exaggerated increase in plasma GH occurred during GTT-2 during pneumococcal sepsis with a peak GH of 20.8 ± 5.9 ng/ml at 30 min as compared by paired t test with the control (GTT-1) of 7.8 ± 2.8 at 30 min (p < 0.05). This hypersecretion of GH during GTT-2 also ...

Purified ovine growth hormone releasing factor: effects on growth hormone secretion and pituitary cyclic nucleotide accumulation.

Growth hormone releasing factor (GRF) was purified from ovine hypothalami on a Sephadex G-25 column. Three fractions from one of the major protein peaks induced the release of radioimmunoassayable growth hormone (GH) from rat pituitaries incubated <i>in vitro</i>, but did not induce any appreciable release of other anterior pituitary hormones. The kinetics of GH release from rat pituitaries included an initial phase with an exponential release of hormones, a subsequent phase with a constant rate of release, and a terminal declining rate of release seen with high GRF doses. The minimally effective dose of this GRF preparation which stimulated GH secretion <i>in vitro</i> contained 1.5 <i>µ</i>g of protein. No change in pituitary cyclic AMP was observed following the exposure of the explants to 3 or 6 <i>µg </i>of GRF for 10 min, but proportional increases in pituitary cyclic GMP were observed at this time period. Although the synthetic decapeptide, Val-His-Leu-Ser-Ala-Glu-Glu-Lys-Glu-Ala, has been shown to deplete bioassayable pituitary GH, it did not stimulate the release of radioimmunoassayable GH nor did it substantially elevate the pituitary cyclic AMP content at the dose levels and time intervals tested. The synthetic decapeptide did not alter pituitary cyclic GMP content after 10 or 60 min of incubation. These observations further substantiate that GRF capable of releasing radioimmunoassayable GH can be isolated from hypothalamic tissue. Preliminary studies of the mechanism of action of GRF indicate that cyclic GMP rather than cyclic AMP plays a central role in mediating the neurohormonal control of GH release.

Solid phase synthesis of growth hormone-release inhibiting factor

The growth hormone-release inhibiting factor (GH-RIF) tetradecapeptide ▪, was prepared by the cyclization of a linear, disulfhydryl peptide intermediate by treatment with potassium ferricyanide. The linear peptide was synthesized by an automated solid-phase technique. The cyclic material inhibited the secretion of radioimmunoassayable growth hormone in vitro at doses as low as 0.1 μg. A high molecular weight compound formed during the cyclization reaction and believed to be predominantly dimer also possessed considerable inhibitory activity.

Pituitary and serum growth hormone during undernutrition and catch-up growth in young rats.

Sprague-Dawley rats at birth were redistributed among nursing mothers, 4 to some, 16 to others. After 21 days, they were studied by sex and by nutritional state using a heterologous radioimmunoassay for growth hormone (GH) content of pituitary gland and plasma. Greater access to milk resulted in mean body weights of males and females of 63 ± 1 g contrasting with 34 ± 2, p < 0.01,and pituitary weights of 3.0 ± 0.07 mg, contrasting with 2.0 ± 0.06, p < 0.01. Better nutrition was attended by plasma GH of 34 ± 14 ng/ml, in contrast to 14 ± 2 ng/ml in the undernourished. There were highly significant differences in total pituitary growth hormone (257 ± 12 μg/gland in well-fed; 88 ± 6 μg/gland in the undernourished). During two weeks of free access to food, the rats from the 16 per mother group grew at 5 times their previous rate (7.5 g per day vs 1.5 g) and nearly overtook the previously better-fed group; plasma growth hormone with dietary repletion was the highest observed, and pituitary weights and growth ho...

Enzymatic synthesis of growth hormone releasing factor (GH-RF) by rat incubates and by extracts of rat and porcine hypothalamic tissue.

SummaryIncubations of rat hypothalamic fragments and of extracts of rat and porcine hypothalamic tissue found previously to synthesize thyrotropin releasing hormone (TRH), luteinizing hormone releasing factor (LRF) and prolactin releasing factor (PRF) have been used to study the biosynthesis of growth hormone releasing factor (GH-RF). To demonstrate synthesis, GH-RF was measured by the elevation of radioimmunoassayable growth hormone in the estrogen-progesterone-reserpinized male rat. GH-RF is synthesized by rat hypothalamic fragments incubated in Krebs-Ringer bicarbonate buffer under an atmosphere of 95% oxygen and 5% carbon dioxide and by extracts of rat and porcine hypothalamic tissue in the presence of amino acids, ATP and Mg2+. This synthesis is not blocked by puromycin, indicating that the synthesizing system may not be ribosomal dependent. Further proof of a nonribosomal mechanism comes from the fact that a ribosomal-free extract also synthesizes GH-RF. From these observations, it may be concluded ...

Alterations of radioimmunoassayable growth hormone and prolactin during hypothroidism.

Induction of hypothyroidism by treatment of rats with propylthiouracil (PTU) or radioactive iodine reduced pituitary and plasma growth hormone concentrations. Radioimmunoassayable pituitary and plasma growth hormone was diminished after three weeks of PTU ingestion. By four weeks, plasma growth hormone had fallen to barely detectable levels in most animals. Pituitary growth hormone concentration reached the lowest value by 40 days and remained depressed throughout 98 days of PTU ingestion. After treatment for 21 days with PTU, rats given tap water had progressively increasing pituitary growth hormone concentrations reaching control levels within 19 days. Rats given PTU and thyroxine injections to prevent hypothyroidism had no appreciable change in pituitary or plasma growth hormone concentrations. Administration of cortisol to chronically hypothyroid animals failed to increase pituitary growth hormone content. When rats bearing the growth hormone— and prolactin— producing tumor MtTWl5 were rendered hypothyroid, plasma and tumor growth hormone contents were substantially reduced. In contrast to the dramatic effects of hypothyroidism on plasma growth hormone concentration, plasma prolactin concentration was altered little by thyroxine deficiency. Although the mean plasma prolactin concentration in hypothyroid male rats was higher than the mean values found in euthyroid animals, the differences were not statistically significant. Pituitary prolactin content was reduced by about 50% in hypothyroid male and female rats indicating a modest reduction in the amount of prolactin stored in the lactotrope. The ability of the transplantable tumor MtTWl5 to produce prolactin was not diminished by the hypothyroid state. In fact, there were increased tumor and plasma prolactin concentrations in MtTWlSbearing hypothyroid rats. In these same animals the pituitary prolactin content was decreased by PTU to levels below those found in euthyroid tumor—bearing rats. These studies suggest that hypothyroidism selectively diminished growth hormone production from the somatotrope. No such direct effect of thyroxine deficiency on prolactin production from the lactotrope was found. The lowered pituitary prolactin storage seen in hypothyroid rats could be the result of an increase in prolactin inhibiting factor activity in these animals. The elevated plasma prolactin found in hypothyroid tumorbearing rats indicates that thyroxine deficiency did not decrease synthesis and secretion of prolactin. In fact, hypothyroidism lead to an apparent increase in prolactin production by lactotropes removed from hvpothalamic control. (Endocrinology92: 487, 1973)

Elevation of Plasma Radioimmunoassayable Growth Hormone in the Rat Induced by Porcine Hypothalamic Extract1

The administration of estradiol benzoate, 50 μg, and progesterone, 25 mg, daily for three days has been found to “sensitize” rats to the growth hormone (GH) releasing activity of hypothalamic extracts. At ten minutes following the injection of 90% methanol extracts of 10 porcine hypothalamic fragments, a significant rise in radioimmunoassayable GH was detected (mean increase in pentobarbital anesthetized rats was 28.5 ng/ml ± 12.3 (SE) VS control of 1.6 ng/ml ± 7.9, p < 0.05). The equivalent of 25 hypothalamic fragments gave a significantly larger response. In a series of animals anesthetized with either pentobarbital or ether, 25 or 10-20 hypothalamic equivalents caused significant elevations of RIA-GH and had no depleting effect on pituitary GH concentration. Without steroid pretreatment, significant effects on plasma GH were not observed even with twenty-five hypothalamic equivalents. In steroid pretreated animals, large doses of vasopressin (5 U) and oxytocin (2 U) also induced GH discharge. TRH at a ...

Effects of pinealectomy, constant light and darkness on growth hormone levels in the pituitary and plasma of the rat.

SUMMARY The effects of constant light, constant darkness, and diurnal lighting, in combination with pinealectomy or sham pinealectomy, on pituitary and plasma concentrations of radio-immunoassayable growth hormone (GH) were investigated in 23- to 24-day-old male rats. In addition, the effects on pituitary, accessory sex organ, testes, adrenal and body weights, as well as tail length, were measured. Three days after operation random groups of pinealectomized and sham-pinealectomized animals were placed in either continuous light, continuous darkness or diurnal light, and killed 28–29 days later. Analysis of the results revealed that the sham-pinealectomized group exposed to constant darkness differed from the other five groups by showing lower pituitary and plasma GH concentrations, body weight, tail length and accessory sex organ weight relative to body weight and testes weight. No statistically significant differences were found in pituitary or adrenal weights. It is concluded that increased pineal function occurring in darkness probably inhibits secretion of GH releasing factor, decreasing pituitary synthesis and release of GH.

Adrenergic and serotoninergic control of growth hormone secretion in adult male rats.

Plasma radioimmunoassayable growth hormone (GH) levels measured in adult male rats anesthetized for 2 hr with urethane, were found to be lower and strikingly more uniform than those of animals decapitated while awake. Rats prepared in this manner, and bearing a cannula chronically implanted in a lateral ventricle of the brain, were used for the determination of the effect, on plasma GH levels, of the intraventricular administration of catecholamines, adrenergic receptor blockers, and serotonin (5- HT). While saline (10 μl/rat) or noradrenaline (1.0 μg/rat) did not induce any significant changes in plasma GH, dopamine (1.0 Mg/rat) significantly reduced plasma GH levels. This dopaminergic inhibition is apparently exerted through hypothalamic beta-adrenergic receptors, since it is abolished by the previous administration of propranolol (5O μg/rat), a specific beta-adrenergic blocker. The injections of 5-HT (1.0 μg/rat) were followed by a highly significant rise of plasma GH; this effect was abolished by the ...

Effects of cold exposure on plasma growth hormone in the adrenalectomized and thyroparathyroidectomized rat.

It has previously been shown that plasma radioimmunoassayable growth hormone (GH) falls following exposure to cold. This response was examined in the normal, adrenalectomized, thyroparathyroidectomized, and adrenalectomized-thyroparathyroid-ectomized rat to determine the effects of the absence of corticosterone (B) and of the thyroid and parathyroid glands on this response. GH decreased from a high prestress level to a low poststress level in both the normal and adrenalectomized animal. The thyropara-thyroidectomized and the adrenalectomized-thyroparathyroidectomized rats had a low prestress GH level, which was not decreased further following cold exposure. The prestress B levels were higher in the thyroparathyroidectomized animals than in the normal. Poststress levels of B were similar in both groups. It appears that an intact thyroid and/or parathyroid gland is necessary to maintain the normal resting plasma levels of GH and B, whereas stress levels do not appear to be affected.

Hypothalamic mediation of growth hormone and adrenal stress response in the squirrel monkey.

Hypothalamic lesions were produced in the squirrel monkey in order to identify the neural pathways by which the emotional stimulus of capture and the stress of ether anesthesia bring about growth hormone (GH) hypersecretion. Plasma GH levels were determined by human GH radioimmunoassay. As an index of pituitary-adrenal activation in these animals, plasma corticoids were measured by competitive binding assay. In unlesioned animals capture led to increased release of GH and to adrenal cortical activation. Chair restraint was followed by continued adrenal cortical hypersecretion unaccompanied Tjy GH discharge, while ether anesthesia induced GH discharge without a significant increase in adrenocortical secretion. Growth hormone responses to capture and ether anesthesia were blocked by relatively small lesions in either anterior or posterior median eminence, while adrenal responses to capture and chair restraint were blocked by lesions in the posterior median eminence in 3 animals. Lesions of the inferior third of the mammillary bodies blocked GH and adrenal responses to capture. GH responses to ether were greatly enhanced in 3 animals with midline optic chiasm lesions and in one animal with a mammillary body lesion, suggesting that structures in these regions may have an inhibiting effect on GH responses. (Endocrinology89: 694, 1971)

Ineffectiveness of hypoglycemia, cold exposure and fasting in stimulating GH secretion in the mouse.

The effects of insulin hypoglycemia, cold exposure and prolonged fasting on plasma and pituitary radioimmunoassayable growth hormone (GH) were studied in the mouse. None of these stimuli, with the exception of fasting for a short interval (16 hr), which induced a slight depletion of pituitary GH content, was capable of increasing plasma or decreasing pituitary GH levels in this animal species. The possible reason for the ineffectiveness of these GHreleasing stimuli in the mouse is discussed. (Endocrinology 88: 345, 1971

Interactions of the pineal gland, blinding, and underfeeding on reproductive organ size and radioimmunoassayable growth hormone.

Restricting male rats to one-half normal food intake from day 25 until day 60 of life significantly delayed all aspects of normal growth that were measured. Body weights were reduced by 50% and pituitary growth hormone (GH) and plasma GH levels were severly reduced compared with corresponding values of <i>ad libitum </i>controls. Underfeeding also partially inhibited testicular and accessory organ growth. If underfeeding was combined with blinding, a more marked inhibition of testicular and accessory organ growth occurred. The effect of combining blinding and underfeeding was more dramatic than the effect of each treatment independently, indicating an accentuation of the response with the combination of treatments. This marked lag in development of the reproductive organs of blinded underfed rats was apparently mediated by the pineal gland, since blinded-pinealectomized underfed rats had reproductive organs that were considerably larger than those of blinded underfed rats.

Pineal regulation of growth hormone synthesis and release in blinded and blinded-anosmic male rats.

Removal of eyes from young male rats led to slight retardation in body weight gain, tibial length, and tail length compared with respective parameters measured in normal rats. Young rats lacking eyes and pineal glands presented body weights, tibial lengths, and tail lengths that approached normal. Pituitary levels of radio-immunoassayable growth hormone (GH) were significantly lower in blinded rats with intact pineal glands compared with levels in normal or blinded-pinealectomized rats. Plasma levels of GH tended to be lower in blinded and blinded- pinealectomized rats relative to normal levels; however, due to the wide range of GH levels in all groups, no statistically significant differences were observed. Blindness and anosmia in young male rats severely retarded body weight gain, tibial length, and tail length relative to respective parameters of normal, blinded, blinded-pinealectomized, and blinded-anosmic-pinealectomized rats. Blinded-anosmic-pineal-ectomized rats grew subnormally, with body weights equal to those of blinded rats. Plasma GH concentrations were low in blinded-anosmic rats, but due to the overlapping values, statistical significance was not attained. In general, the size of reproductive organs correlated well with inhibition of body growth, being moderately smaller in blinded rats and markedly so in blinded-anosmic rats. These differences in the size of the reproductive organs were not observed if pinealectomy was performed simultaneously with blinding and olfactory bulb removal. It is concluded that, in young male rats that are deprived of light by blinding, there is a dramatic inhibition of GH production and release by the pituitary gland. This response, like the response of the reproductive organs, is enhanced when rats are deprived of both smell and light. These phenomena only occur in the presence of the pineal gland. Therefore, the pineal gland plays an important role in these processes. It is further concluded that anosmia or perhaps non-specific surgical stress may alter GH synthesis and/or release from the pituitary gland.